Novel carbonyl reductase, gene thereof and method of using the same

ABSTRACT

The present invention provides a novel polypeptide producing (S)-N-benzyl-3-pyrrolidinol, a DNA coding for it and a method of using them.  
     A polypeptide having the following physicochemical properties (1) to (5):  
     (1) Action: It asymmetrically reduces N-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol with NADPH as a coenzyme;  
     (2) Optimum action pH: 4.5 to 5.5;  
     (3) Optimum action temperature: 40° C. to 45° C.;  
     (4) Molecular weight: About 29,000 as determined by gel filtration analysis, about 35,000 as determined by SDS-polyacrylamide gel electrophoresis analysis;  
     (5) Inhibitor: It is inhibited by the divalent copper ion.  
     Further, a polypeptide having the amino acid sequence shown under SEQ ID NO:1 in the sequence listing; or  
     a polypeptide having an amino acid sequence obtainable from the amino acid sequence shown under SEQ ID NO:1 in the sequence listing by substitution, insertion, deletion and/or addition of one or more amino acids and  
     having enzyme activity in asymmetrically reducing N-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol.

TECHNICAL FIELD

[0001] The present invention relates to a novel polypeptide, a genecoding for the polypeptide, an expression vector for the expression ofthe polypeptide, a transformant obtained by transformation of a hostusing the expression vector, and a production method of a compounduseful as a material for the synthesis of medicinal and other compoundsusing the above transformant.

[0002] In more detail, the invention relates to a polypeptide isolatedfrom a microorganism having enzyme activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol andhaving such enzyme activity, a DNA coding for the polypeptide, anexpression vector containing the DNA, and a transformant obtained bytransformation using the expression vector. The present invention alsorelates to a production method of (S)-N-benzyl-3-pyrrolidinol.

[0003] (S)-N-Benzyl-3-pyrrolidinol is a compound useful as anintermediate for the synthesis of medicinal compounds such as β-lactamantibiotics and dihydropyridine compounds.

BACKGROUND ART

[0004] Known as the production method of optically active(S)-N-benzyl-3-pyrrolidinol are the method which comprises synthesizingfrom an optically active compound and the method which comprisescarrying out asymmetric synthesis or optical resolution starting with aprochiral compound. As such a method, JP-A-06-141876 discloses aproduction method of optically active N-benzyl-3-pyrrolidinol whichcomprises stereoselectively reducing N-benzyl-3-pyrrolidinone in thepresence of an enzyme having activity in stereoselectively reducing thisN-benzyl-3-pyrrolidinone. Further, JP-A-10-150997 discloses a productionmethod of optically active N-benzyl-3-pyrrolidinol which comprisestreating N-benzyl-3-pyrrolidinone with a cell or a culture of amicroorganism or a treated product thereof. However, these methods arelow in attainable substrate concentration and in conversion from thesubstrate to the product, hence cannot be put to practical use.

SUMMARY OF THE INVENTION

[0005] The present inventors found a microorganism-derived polypeptidewhich asymmetrically reduces N-benzyl-3-pyrrolidinone to produce(S)-N-benzyl-3-pyrrolidinol and found that (S)-N-benzyl-3-pyrrolidinolcan be produced efficiently, and have now completed the presentinvention.

[0006] It is an object of the present invention to provide a polypeptidecapable of asymmetrically reducing N-benzyl-3-pyrrolidinone to produce(S)-N-benzyl-3-pyrrolidinol. Another object of the invention is toproduce that polypeptide efficiently utilizing the gene recombinationtechnology. A further object of the invention is to provide atransformant capable of simultaneously producing, at high levels, thatpolypeptide and a polypeptide having glucose dehydrogenase activity and,further, provide a practical production method of(S)-N-benzyl-3-pyrrolidinol using that transformant.

[0007] Thus, the present invention comprises a polypeptide having thefollowing physicochemical properties (1) to (5):

[0008] (1) Action: It asymmetrically reduces N-benzyl-3-pyrrolidinone toproduce (S)-N-benzyl-3-pyrrolidinol with NADPH as a coenzyme;

[0009] (2) Optimum action pH: 4.5 to 5.5;

[0010] (3) Optimum action temperature: 40° C. to 45° C.;

[0011] (4) Molecular weight: About 29,000 as determined by gelfiltration analysis, about 35,000 as determined by SDS-polyacrylamidegel electrophoresis analysis;

[0012] (5) Inhibitor: It is inhibited by the divalent copper ion.

[0013] Further, the present invention is a polypeptide described in thefollowing (a) or (b):

[0014] (a) A polypeptide having the amino acid sequence shown under SEQID NO:1 in the sequence listing;

[0015] (b) A polypeptide having an amino acid sequence obtainable fromthe amino acid sequence shown under SEQ ID NO:1 in the sequence listingby substitution, insertion, deletion and/or addition of one or moreamino acids and

[0016] having enzyme activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol.

[0017] Furthermore, the present invention comprises DNAs coding forthese polypeptides. Or, it also comprises a DNA coding for a polypeptidehaving enzyme activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol, andhybridizing with a DNA having a nucleotide sequence shown under SEQ IDNO:2 in the sequence listing under stringent conditions, or a DNA codingfor a polypeptide having enzyme activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol, andhaving at least 60% sequence identity with a nucleotide sequence shownunder SEQ ID NO:2 in the sequence listing.

[0018] Furthermore, it comprises an expression vector containing any ofthese DNAs and a transformant containing such expression vector.

[0019] The present invention also comprises a production method of(S)-N-benzyl-3-pyrrolidinol comprising

[0020] a step of reacting such transformant and/or a treated productthereof with N-benzyl-3-pyrrolidinone, and

[0021] a step of harvesting the thus-produced(S)-N-benzyl-3-pyrrolidinol.

DETAILED DISCLOSURE OF THE INVENTION

[0022] In the following, the present invention is described in detail.

[0023] First, the polypeptide of the invention is described.

[0024] The polypeptide of the invention has enzyme activity inasymmetrically reducing N-benzyl-3-pyrrolidinone represented by theformula (I) shown below to produce (S)-N-benzyl-3-pyrrolidinolrepresented by the formula (II) shown below.

[0025] As such polypeptide, there may be mentioned an enzyme having thefollowing physicochemical properties (1) to (5).

[0026] (1) Action: It asymmetrically reduces N-benzyl-3-pyrrolidinone toproduce (S)-N-benzyl-3-pyrrolidinol with NADPH as a coenzyme;

[0027] (2) Optimum action pH: 4.5 to 5.5;

[0028] (3) Optimum action temperature: 40° C. to 45° C.;

[0029] (4) Molecular weight: About 29,000 as determined by gelfiltration analysis, about 35,000 as determined by SDS-polyacrylamidegel electrophoresis analysis;

[0030] (5) Inhibitor: It is inhibited by the divalent copper ion.

[0031] In the present invention, the enzyme activity of the polypeptideis determined by adding the substrate N-benzyl-3-pyrrolidinone (1 mM),the coenzyme NADPH (0.167 mM) and the enzyme to 100 mM phosphate buffer(pH 6.5) and measuring the decrease in absorbance at the wavelength 340nm at 30° C.

[0032] The optimum action pH and optimum action temperature of thepeptide are determined, for example by varying the reaction pH orreaction temperature in the above reducing activity measurement systemand measuring the reducing activity.

[0033] The gel filtration analysis-based molecular weight of the peptideis determined by calculation from the elution time relative to those ofreference proteins in gel filtration. The SDS-polyacrylamide gelelectrophoresis-based molecular weight is determined by calculation fromthe mobility relative to those of reference proteins inSDS-polyacrylamide gel electrophoresis.

[0034] The inhibitors are determined, for example by adding variouscompounds to the above reducing activity measurement system andmeasuring the reducing activity of each compound.

[0035] The polypeptide of the invention can be obtained from amicroorganism having an activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol. Thus,the microorganism to be used as the source of the polypeptide is notparticularly restricted but includes, for example, microorganismsbelonging to the genus Micrococcus, among which the strain Micrococcusluteus IFO 13867 is particularly preferred. The microorganism producingthe polypeptide of the invention may be a wild strain or a mutant. Or, amicroorganism obtainable by cell fusion or by such a genetic method asgene manipulation may also be used. A gene manipulated microorganismcapable of producing the polypeptide of the invention can be obtained bya method comprising a step of isolating and/or purifying such enzyme anddetermining a part or the whole of the amino acid sequence thereof, astep of determining a nucleotide sequence coding for the enzyme based onthat amino acid sequence, a step of obtaining a nucleotide sequencecoding for the enzyme based on that amino acid sequence, and a step ofobtaining a recombinant microorganism by introducing that nucleotidesequence into another microorganism.

[0036] As for the culture medium for the microorganism producing thepolypeptide of the invention, ordinary liquid culture medium containingcarbon sources, nitrogen sources, inorganic salts, organic nutrients andso on can be used provided that the microorganism can grow thereon.

[0037] The term “culture of the microorganism” as used in thisspecification means cells of the microorganism or a culture fluidcontaining such cells. The “treated product thereof” means an extract orpurified product obtained from the cells of the microorganism or aculture fluid containing such cells by extraction, purification or someother treatment.

[0038] The polypeptide of the invention can be purified from themicroorganism producing that polypeptide in the conventional manner. Forexample, cells of the microorganism are cultured on an appropriatemedium, and cells are harvested from the culture fluid bycentrifugation. The cells obtained are disrupted using a sonicator, forinstance, and the cell residue is removed by centrifugation to give acell-free extract. The polypeptide can be purified from this cell-freeextract by applying, singly or in combination, such techniques assalting out (e.g. ammonium sulfate precipitation, sodium phosphateprecipitation), solvent precipitation (protein fractionationprecipitation using acetone, ethanol or the like), dialysis, gelfiltration, ion exchange, column chromatography such as reversed phaseand ultrafiltration.

[0039] The polypeptide of the invention may be a natural enzyme obtainedfrom a microorganism as mentioned above or may be a recombinant enzyme.As a natural enzyme, there may be mentioned a polypeptide having theamino acid sequence shown under SEQ ID NO:1 in the sequence listing.

[0040] The polypeptide of the invention may also be a polypeptide havingan amino acid sequence obtainable from the amino acid sequence shownunder SEQ ID NO:1 in the sequence listing by substitution, insertion,deletion and/or addition of one or more amino acids and having enzymeactivity in asymmetrically reducing N-benzyl-3-pyrrolidinone to produce(S)-N-benzyl-3-pyrrolidinol.

[0041] Such polypeptide can be prepared from the polypeptide having theamino acid sequence shown under SEQ ID NO:1 in the sequence listing bysuch a known method as described in Current Protocols in MolecularBiology (John Wiley and Sons, Inc., 1989).

[0042] The phrase “having enzyme activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol” is usedherein to indicate that when the polypeptide in question is subjected toreaction with N-benzyl-3-pyrrolidinone under the above-mentionedreducing activity measurement conditions, (S)-N-benzyl-3-pyrrolidinol isproduced in a yield not less than 10%, preferably not less than 40%,more preferably not less than 60%, as compared with the case where thepolypeptide having the amino acid sequence shown under SEQ ID NO:1 inthe sequence listing is used.

[0043] The DNA of the invention is described in the following.

[0044] The DNA of the invention may be any DNA coding for such apolypeptide as mentioned above. It may be a DNA having the nucleotidesequence shown under SEQ ID NO:2 in the sequence listing, or a DNAcoding for a polypeptide having enzyme activity in asymmetricallyreducing N-benzyl-3-pyrrolidinone to produce(S)-N-benzyl-3-pyrrolidinol, and hybridizing with the DNA having thenucleotide sequence shown under SEQ ID NO:2 in the sequence listingunder stringent conditions.

[0045] The term “DNA hybridizing with the DNA having the nucleotidesequence shown under SEQ ID NO:2 in the sequence listing under stringentconditions” means a DNA obtainable by the technique of colonyhybridization, plaque hybridization or southern hybridization, using theDNA having the nucleotide sequence shown under SEQ ID NO:2 in thesequence listing as a probe. More specifically, there may be mentioned aDNA identified by carrying out hybridization using a filter with thecolony or plaque-derived DNA immobilized thereon, at 65° C. in thepresence of 0.7 to 1.0 M NaCl, and then washing the filter with a 0.1-to 2-fold concentrated SSC solution (1-fold concentrated SSC solutioncomprising 150 mm sodium chloride and 15 mM sodium citrate) at 65° C.

[0046] The hybridization can be carried out according to the methoddescribed in Molecular Cloning, A laboratory manual, second edition(Cold Spring Harbor Laboratory Press, 1989) or elsewhere.

[0047] The DNA of the invention may be a DNA coding for a polypeptidehaving enzyme activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol andhaving at least 60% sequence identity, preferably at least 80% sequenceidentity, more preferably at least 90% sequence identity, still morepreferably at least 95% sequence identity, most preferably at least 99%sequence identity, with the nucleotide sequence shown under SEQ ID NO:2in the sequence listing.

[0048] The term “sequence identity” means that the two nucleotidesequences under comparison are identical with each other, and thepercentage (%) of sequence identity between two nucleotide sequencesunder comparison is calculated by optimally arranging the two nucleotidesequences under comparison, counting those positions at which the samenucleotide (e.g. A, T, C, G, U or I) appears in both the sequences,dividing the thus-found number of conforming positions by the totalnumber of bases under comparison and multiplying the quotient by 100.The sequence identity can be calculated using the following tools forsequence analysis: Unix Base GCG Wisconsin Package (Program Manual forthe Wisconsin Package, Version 8, September 1994, Genetics ComputerGroup, 575 Science Drive Madison, Wis., USA 53711; Rice, P. (1996)Program Manual for EGCG Package, Peter Rice, The Sanger Centre, HinxtonHall, Cambridge, CB10 1RQ, England) and the ExPASy World Wide WebMolecular Biology Server (Geneva University Hospital and University ofGeneva, Geneva, Switzerland).

[0049] The DNA of the invention can be obtained from a microorganismhaving enzyme activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol. As themicroorganism, there may be mentioned, for example, microorganismsbelonging to the genus Micrococcus and, as a particularly preferredstrain, there may be mentioned the strain Micrococcus luteus IFO 13867.

[0050] In the following, an embodiment of the method of obtaining theDNA of the invention from a microorganism having enzyme activity inasymmetrically reducing N-benzyl-3-pyrrolidinone to produce(S)-N-benzyl-3-pyrrolidinol is described.

[0051] First, partial amino acid sequence of the purified polypeptideand of peptide fragments obtainable by digestion of that polypeptidewith an appropriate endopeptidase are determined by the Edman technique.DNA primers are synthesized based on the thus-obtained amino acidsequence information. Then, the chromosomal DNA of the microorganism isprepared from that microorganism, which is the source of the above DNA,by a conventional method of DNA isolation, for example by the method ofMurray et al. (Nucl., Acids Res. 8:4321-4325 (1980)). Using the aboveDNA primers, PCR is carried out with the chromosomal DNA as the templateto amplify part of the polypeptide gene. Further, DNA probes areprepared by labeling part of the thus-amplified polypeptide gene byconventional methods, for example by the random primer labeling method(Anal. Biochem., 132, 6 (1983)). The chromosomal DNA of themicroorganism is cleaved with an appropriate restriction enzyme, therestriction enzyme cleaved fragments are inserted into a vector and theresulting vectors are introduced into appropriate host cells to therebyconstruct a DNA library of the microbial chromosome. Screening of thisDNA library is carried out by the colony hybridization, plaquehybridization or like method using the above DNA probes, whereby a DNAcontaining the polypeptide gene can be obtained. The nucleotide sequenceof the thus-obtained DNA fragment containing the polypeptide gene can bedetermined by the dideoxy sequencing method or dideoxy chain terminationmethod, or the like. For example, this can be carried out using the ABIPRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (product ofPerkin-Elmer) and the ABI 373A DNA Sequencer (product of Perkin-Elmer).

[0052] The expression vector and transformant of the present inventionare now described.

[0053] The enzyme gene can be expressed in the transformant which isobtainable by inserting the DNA of the present invention to a vector andintroducing the vector into a host. The vector to be used for thispurpose may be any of those capable of expressing the enzyme gene inappropriate hosts. As such vector, there may be mentioned, for example,a plasmid vector, phage vector, cosmid vector, etc. It may be a shuttlevector capable of gene exchange between different host strains.Generally, such vector comprises such a regulatory factor as the lac UV5promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tuf Bpromoter, rec A promoter or pL promoter, and is suitably used as anexpression vector containing an expression unit operatively connected tothe DNA of the invention.

[0054] The term “regulatory factor” as used herein means a functionalpromoter and a nucleotide sequence having arbitrary relatedtranscription elements (e.g. enhancer, CCAAT box, TATA box, SPI site,etc.).

[0055] The term “operatively connected” as used herein means that theDNA and various regulatory elements, such as promoter and enhancer,which regulate the expression thereof are joined together each in astate operative in a host so that the gene can be expressed. It is wellknown to the artisan that the type and species of the regulatory factormay vary according to the host.

[0056] As the host into which an expression vector containing the DNA ofthe invention is to be introduced, there may be mentioned bacteria,yeasts, filamentous fungi, plant cells and animal cells, for instance.Escherichia coli is preferred, however. The DNA of the invention can beintroduced into a host in the conventional manner. When Escherichia coliis used as the host, the DNA of the invention can be introduced into thesame by the calcium chloride method, for instance.

[0057] For producing (S)-N-benzyl-3-pyrrolidinol by asymmetricallyreducing N-benzyl-3-pyrrolidinone using the DNA of the invention, acoenzyme such as NAPDH or NADH is required. However, by carrying out thereaction using an enzyme capable of converting the coenzyme oxidized toits reduced form (hereinafter referred to as coenzyme regeneratingability) together with a substrate thereof, namely combining a coenzymeregeneration system with the polypeptide of the invention, it ispossible to markedly reduce the consumption of the coenzyme, which isexpensive. Usable as the enzyme having coenzyme regenerating abilityare, for example, hydrogenase, formate dehydrogenase, alcoholdehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenaseand glucose dehydrogenase. Glucose dehydrogenase is suitably used.

[0058] When a transformant containing both the DNA of the invention anda DNA coding for a polypeptide having glucose dehydrogenase activity isused, the above reaction can be carried out efficiently withoutseparately preparing an enzyme having coenzyme regenerating ability andadding the same to the reaction system, although such reaction may alsobe carried out by adding a coenzyme regeneration system to theasymmetric reduction reaction system. Such transformant can be obtainedby inserting the DNA of the invention and a DNA coding for a polypeptidehaving glucose dehydrogenase activity into the same vector andintroducing this into a host, or by inserting these two DNAsrespectively into two different vectors belonging to incompatible groupsand introducing these into the same host. Thus, a transformantcontaining an expression vector comprising the DNA of the invention andthe DNA coding for a polypeptide having glucose dehydrogenase activity,or a transformant containing both a first expression vector containingthe DNA of the invention and an expression vector containing the DNAcoding for a polypeptide having glucose dehydrogenase activity can beused. As for the polypeptide having glucose dehydrogenase activity,Bacillus megaterium-derived one is preferred.

[0059] The glucose dehydrogenase activity in the transformant isdetermined by adding the substrate glucose (0.1 M), the coenzyme NADP (2mM) and the enzyme to 1 M Tris hydrochloride buffer (pH 8.0) andmeasuring the increase in absorbance at the wavelength 340 nm at 25° C.

[0060] Now, a production of (S)-N-benzyl-3-pyrrolidinol using thetransformant of the invention is described.

[0061] Such production method comprises a step of reacting the abovetransformant and/or a treated product thereof withN-benzyl-3-pyrrolidinone and a step of harvesting the thus-produced(S)-N-benzyl-3-pyrrolidinol.

[0062] In the following, this method is more specifically described.First, the substrate N-benzyl-3-pyrrolidinone, NADPH or a like coenzyme,and a culture of the above transformant and/or a treated productthereof, are added to an appropriate solvent, and the reaction isallowed to proceed under stirring with the pH adjusted. This reaction iscarried out at a temperature of 10° C. to 70° C., and the pH ismaintained at 4 to 10 during the reaction. The reaction can be carriedout batchwise or continuously. In the batchwise, the reaction substrateis added to a charge concentration of 0.1% to 70% (w/v). The treatedproduct of the transformant so referred to herein means, for example, acrude extract, cultured cells, lyophilized organism bodies,acetone-dried organism bodies, a disruption product derived therefromand the like. Further, these can be used in the form of the enzymeitself or cells as such immobilized by known means. This reaction ispreferably carried out in the presence of a coenzyme regenerationsystem. For example, when, in carrying out this reaction, a transformantcapable of producing both the polypeptide of the invention and glucosedehydrogenase is used, it is made possible to markedly reduce theconsumption of the coenzyme by further adding glucose to the reactionsystem.

[0063] The (S)-N-benzyl-3-pyrrolidinol produced by the reaction can beharvested by a conventional method. For example, the suspended matter,such as cells, is removed, if necessary, by such treatment ascentrifugation or filtration, the reaction solution is made basic byaddition of sodium hydroxide or the like and extracted with an organicsolvent such as ethyl acetate or toluene, and the organic solvent isthen removed under reduced pressure. The product can be purified byfurther treatment such as distillation or chromatography and so on.

[0064] N-Benzyl-3-pyrrolidinone, which is to serve as the substrate inthe reaction, can be prepared, for example, by the method described inJP-A-54-16466.

[0065] The quantities of N-benzyl-3-pyrrolidinone and(S)-N-benzyl-3-pyrrolidinol can be determined by gas chromatography(column: Uniport B 10% PEG-20M (3.0 mm ID×1.0 m), column temperature:200° C., carrier gas: nitrogen, detection: FID). The optical purity of(S)-N-benzyl-3-pyrrolidinol can be measured by high performance liquidchromatography (column: Chiralcel OB (product of Daicel ChemicalIndustries), eluent: n-hexane/isopropanol/diethylamine=950/50/1, flowrate: 1 ml/min, detection: 254 nm).

[0066] Thus, according to the present invention, it is possible toefficiently produce the polypeptide included in the present inventionand, by utilizing the same, an advantageous, production method of(S)-N-benzyl-3-pyrrolidinol is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0067]FIG. 1 is a chart of the nucleotide sequence of the DNA asdetermined in Example 3 and the amino acid sequence deduced therefrom.

[0068]FIG. 2 is a chart of the method of constructing the recombinantplasmid pTSBG1 of Example 7 and the structure thereof.

BEST MODES FOR CARRYING OUT THE INVENTION

[0069] The following Examples illustrate the present invention indetail. They are, however, by no means limitative of the scope of thepresent invention.

[0070] Detailed procedures and so on concerning the recombinant DNAtechnology used in the following Examples are described in the followingliteratures.

[0071] Molecular Cloning, 2nd Edition (Cold Spring Harbor LaboratoryPress, 1989);

[0072] Current Protocols in Molecular Biology (Greene PublishingAssociates and Wiley-Interscience).

EXAMPLE 1 Purification of Enzyme

[0073] An enzyme having the activity in asymmetrically reducingN-benzyl-3-pyrrolidinone to produce (S)-N-benzyl-3-pyrrolidinol wasuniformly purified from the strain Micrococcus luteus IFO 13867 in thefollowing manner.

[0074] (Cultivation of the Strain Micrococcus luteus IFO 13867)

[0075] A liquid medium (400 mL) having the following composition wasprepared in each 2-L Sakaguchi flask and steam-sterilized at 120° C. for20 minutes. Medium composition: Trypton 1.6% (w/v) Yeast extract 1.0%(w/v) NaCl 0.5% (w/v) Tap water pH 7.0

[0076] This medium was inoculated with 1 ml of a culture fluid of thestrain Micrococcus luteus IFO 13867 prepared in advance by preculture inthe same medium, and shake culture was carried out at 30° C. for 50hours.

[0077] (Preparation of Cell-Free Extract)

[0078] Cells were collected by centrifugation from the above culturefluid (2 L) and washed with physiological saline. Thus, 42 g of wetcells of the above strain were obtained. These wet cells were suspendedin 170 mL of 100 mM phosphate buffer (pH 7.0), then 2-mercaptoethanoland phenylmethylsulfonyl fluoride were added to respective finalconcentrations of 5 mM and 0.1 mM, and the cells were ultrasonicallydisrupted using SONIFIRE 250 (product of BRANSON). The cell residueswere removed from the disrupted cell disruption by centrifugation,whereby 180 mL of a cell-free extract was obtained.

[0079] (Ammonium Sulfate Fractionation)

[0080] Ammonium sulfate was added to and dissolved in the cell-freeextract obtained in the above manner to attain 40% saturation and theresulting precipitate was removed by centrifugation (during thisprocedure, the pH of the cell-free extract was maintained at 7.0 usingaqueous ammonia). While the pH was maintained at 7.0 in the same manneras in the above procedure, ammonium sulfate was further added to anddissolved in this centrifugation supernatant to attain 65% saturation,and the resulting precipitate was collected by centrifugation. Thisprecipitate was dissolved in 10 mM phosphate buffer (pH 7.0) containing5 mM 2-mercaptoethanol, and the solution was dialyzed overnight usingthe same buffer.

[0081] (Phenyl Sepharose Column Chromatography)

[0082] Ammonium sulfate was dissolved in the crude enzyme solutionobtained in the above manner to a final concentration of 1 M (while thepH of the crude enzyme solution was maintained at 7.0 using aqueousammonia), and the solution was applied to a Phenyl sepharose CL-4B(product of Pharmacia Biotech) column (130 mL) equilibrated in advancewith 10 mM phosphate buffer (pH 7.0) containing 5 mM 2-mercaptoethanoland 1 M ammonium sulfate so as to adsorb the enzyme. After washing thecolumn with the same buffer, the active fraction was eluted with alinear gradient of ammonium sulfate (from 1 M to 0 M). The activefraction was collected and dialyzed overnight using 10 mM phosphatebuffer (pH 7.0) containing 5 mM 2-mercaptoethanol.

[0083] (DEAE Sepharose Column Chromatography)

[0084] The crude enzyme solution obtained in the above manner wasapplied to a DEAE sepharose CL-4B (product of Pharmacia Biotech) column(20 mL) equilibrated in advance with 10 mM phosphate buffer (pH 7.0)containing 5 mM 2-mercaptoethanol so as to adsorb the enzyme. Afterwashing the column with the same buffer, the active fraction was elutedwith a linear gradient of NaCl (from 0 M to 1.0 M). The active fractionwas collected and dialyzed overnight using 10 mM phosphate buffer (pH7.0) containing 5 mM 2-mercaptoethanol.

[0085] (Blue Sepharose Column Chromatography)

[0086] The crude enzyme solution obtained in the above manner wasapplied to a Blue Sepharose CL-6B (product of Pharmacia Biotech) column(10 ml) equilibrated in advance with 20 mM phosphate buffer (pH 6.0)containing 5 mM 2-mercaptoethanol so as to adsorb the enzyme. Afterwashing the column with the same buffer, the active fraction was elutedwith a linear gradient of NaCl (from 0 M to 0.5 M). The active fractionwas collected and dialyzed overnight using 10 mM phosphate buffer (pH7.0) containing 5 mM 2-mercaptoethanol.

[0087] (Gel Filtration)

[0088] The crude enzyme solution obtained in the above manner wasapplied to a TSK-GEL G3000 SWXL column (product of Tosoh) equilibratedin advance with 100 mM phosphate buffer (pH 7.0) containing 5 mM2-mercaptoethanol and 100 mM sodium sulfate, and the active fraction waseluted with the same buffer. The active fraction was collected anddialyzed overnight using 10 mM phosphate buffer (pH 7.0) containing 5 mM2-mercaptoethanol to give an electrophoretically uniform, purifiedenzyme preparation. Hereinafter, this enzyme is referred to as BRD.

EXAMPLE 2 Measurements of Enzyme Properties

[0089] The enzyme obtained was examined for its enzymologicalproperties. The enzyme activity was. determined basically by adding thesubstrate N-benzyl-3-pyrrolidinone (1 mM), the coenzyme NADPH (0.167 mM)and the enzyme to 100 mM phosphate buffer (pH 6.5), allowing thereaction to proceed at 30° C. for 1 minute and measuring the decrease inabsorbance at the wavelength 340 nm.

[0090] (1) Action:

[0091] It acted on N-benzyl-3-pyrrolidinone with NADPH as the coenzymeand produced (S)-N-benzyl-3-pyrrolidinol with an optical purity of notless than 99% ee.

[0092] (2) Optimum Action pH:

[0093] The enzyme activity was measured by the above method within thepH range of 4.0 to 7.0 using phosphate buffer and acetate buffer as abuffer. As a result, the optimum pH for the action onN-benzyl-3-pyrrolidinone was found to be 4.5 to 5.5.

[0094] (3) Optimum Action Temperature:

[0095] The enzyme activity against the substrate N-benzyl-3-pyrrolidinonexerted for one minute of the reaction was measured within thetemperature range of 20° C. to 60° C. As a result, the optimumtemperature was found to be 40° C. to 45° C.

[0096] (4) Molecular Weight:

[0097] The molecular weight of this enzyme was determined by gelfiltration using a TSK-GEL G3000 SWXL column (product of Tosoh) and, asthe eluent, 100 mM phosphate buffer (pH 7.0) containing 5 mM2-mercaptoethanol and 100 mM sodium sulfate. The molecular weight of thesubunit of the enzyme was calculated from the relative mobility relativeto reference proteins in SDS-polyacrylamide gel electrophoresis. As aresult, the molecular weight of the enzyme was found to be about 29,000as determined by gel filtration analysis or about 35,000 as determinedby SDS-polyacrylamide gel electrophoretic analysis.

[0098] (5) Inhibitors:

[0099] The reaction was repeated with the addition of various metal ionsand inhibitors shown in Table 1 and, with the activity without additionbeing taken as 100%, the relative activities upon addition thereof wereexamined. As shown in Table 1, the enzyme was inhibited by the divalentcopper ion. TABLE 1 Addition level Relative activity Compound (mM) (%)None — 100 CoCl₂ 1 99 CuSO₄ 0.1 6 1 5 ZnSO₄ 1 99 MnCl₂ 1 89 MgSO₄ 1 991,10-Phenanthroline 1 90 5,5-Diphenylhydantoin 0.5 99 EDTA 1 88 PMSF 189 PCMB 0.1 78 DTNB 0.01 93 Iodoacetic acid 1 89 NEM 1 94 Quercetin 0.0194

EXAMPLE 3 Cloning of BRD Gene

[0100] (Preparation of Synthetic Oligonucleotide Probes)

[0101] The purified BRD obtained in Example 1 was digested with bovinepancreas-derived trypsin (product of Wako Pure Chemical Industries), andthe amino acid sequences of peptide fragments obtained were determinedusing ABI 492 model protein sequencer (product of Perkin Elmer). Basedon this amino acid sequence, two DNA primers shown under SEQ ID NO:3 andSEQ ID NO:4 in the sequence listing were synthesized in the conventionalmanner.

[0102] (Amplification of BRD Gene by PCR)

[0103] The chromosomal DNA was extracted from cultured cells of thestrain Micrococcus luteus IFO 13867 by the method of Murray et al.(Nucl., Acids Res. 8:4321-4325 (1980)). Then, using the DNA primersprepared as mentioned above, PCR was carried out with the chromosomalDNA obtained as the template, whereupon a DNA fragment (about 250 bp)supposed to be part of the BRD gene was amplified.

[0104] (Construction of Chromosomal DNA Library)

[0105] The chromosomal DNA of the strain Micrococcus luteus IFO 13867was completely digested with the restriction enzyme BamHI, followed byseparation by agarose gel electrophoresis. Then, using the DNA fragmentobtained in the above manner (about 250 bp) as the probe, the digest ofthe chromosomal DNA was analyzed by the Southern method (J. Mol. Biol.,98, 503 (1975)) (the labeling of the DNA probe and detection thereofbeing carried out using the Gene Images labeling/detection system(product of Amersham)). As a result, a DNA fragment of about 4.5 kb wasfound to hybridize with the above DNA probe.

[0106] Therefore, the above digest was subjected to separation byagarose gel electrophoresis, and 4.3 kb to 6.2 kb DNA fragments wererecovered. These DNA fragments were inserted into the vector plasmidpUC19 (product of Takara Shuzo) at the BamHI site thereof, followed byintroduction into the strain Escherichia coli JM109 (product of TakaraShuzo). A chromosomal DNA library of this strain was thus constructed.

[0107] (Screening of the Chromosomal DNA Library)

[0108] Using the DNA fragment obtained in the above manner as the probe,the chromosomal DNA library constructed in the above manner wassubjected to screening by the colony hybridization method (the labelingof the DNA probe and detection thereof being carried out using the GeneImages labeling/detection system (product of Amersham) and theexperimental procedure being performed according to the manual attachedto the system). As a result, one positive colony was obtained.Therefore, a recombinant plasmid pUC-BB, produced by insertion of theDNA (about 4.5 kb) obtained from this positive colony was selected as aBRD gene-containing chromosomal DNA clone.

[0109] (Determination of Nucleotide Sequence)

[0110] The recombinant plasmid pUC-BB obtained in the above manner wastreated with various restriction enzymes and the resulting digestionfragments were analyzed, and a restriction enzyme cleavage map thereofwas prepared. Then, recombinant plasmids were constructed by insertingvarious DNA fragments obtained on the occasion of the above analysisinto pUC19 at the multicloning site thereof. Using these recombinantplasmids, the nucleotide sequences of each inserted fragment wasanalyzed using an ABI PRISM Dye Terminator Cycle Sequencing ReadyReaction Kit (product of Perkin Elmer) and ABI 373A DNA Sequencer(product of Perkin Elmer), and the nucleotide sequence of a DNA fragment(about 1.4 kb) supposed to contain the target gene was determined. Thatnucleotide sequence is shown in FIG. 1. As for the structural geneportion in this nucleotide sequence, the amino acid sequence deducedfrom that nucleotide sequence is shown below the nucleotide sequence inFIG. 1. As a result of comparison of this amino acid sequence with thepartial amino acid sequence of digested fragments of purified BRD bytrypsin, the whole partial amino acid sequence of purified BRD was foundto exist in the amino acid sequence deduced from the nucleotide sequenceand to be quite identical in that portion (the underlined amino acidsequence in FIG. 1). Thus, that gene was judged to be the BRD gene.

EXAMPLE 4 Construction of BRD Gene-Containing Recombinant Plasmid

[0111] A double-stranded DNA resulting from addition of an NdeI site tothe initiation codon portion of the structural gene of BRD and furtheraddition, just behind the 3′ terminus thereof, of a termination codon(TAA) and an EcoRI cleavage point was obtained in the following manner.Based on the nucleotide sequence determined in Example 3, an N-terminalDNA primer with an NdeI site added to the initiation codon part of theBRD gene and a C-terminal DNA primer with a termination codon (TAA) andan EcoRI site added just behind the 3′ terminus of the same gene weresynthesized. The nucleotide sequences of these two primers are shownunder SEQ ID NO:5 and SEQ ID NO:6 in the sequence listing. Using thesetwo synthetic DNA primers, together with the plasmid pUC-BB obtained inExample 3 as the template, a double-stranded DNA was amplified by PCR.The DNA fragment obtained was digested with NdeI and EcoRI, and theresulting fragment was inserted into the plasmid pUCNT (WO 94/03613) atthe NdeI-EcoRI site downstream of the lac promoter to give a recombinantplasmid pNTBR.

EXAMPLE 5 Addition of Shine-Dalgarno Sequence to a Site Upstream of theBRD Gene

[0112] For attaining high level expression of the BRD gene inEscherichia coli, a plasmid was obtained from the plasmid pNTBR preparedin Example 4 by newly adding the E.coli-derived Shine-Dalgarno sequence(9 bases) to a site upstream of the initiation codon of the same gene,as follows. First, a plasmid pUCT was constructed by converting G in theNdeI site of the E.coli expression vector pUCNT used in Example 4 to Tby the PCR method. Then, an N-terminal DNA primer resulting fromaddition of the E.coli-derived Shine-Dalgarno sequence (9 bases) at 5bases upstream of the initiation codon of the BRD gene shown under SEQID NO:2 in the sequence listing and further addition of an EcoRI site atjust before the Shine-Dalgarno sequence and a C-terminal DNA primerresulting from addition of a SacI site just behind the 3′ terminus ofthe same gene were synthesized in the conventional manner. Thenucleotide sequences of these two primers are shown under SEQ ID NO:7and SEQ ID NO:8 in the sequence listing. Using these two DNA primers,with the plasmid pNTBR constructed in Example 4 as the template, adouble-stranded DNA was synthesized by PCR. The DNA fragment obtainedwas digested with EcoRI and SacI, and the resulting fragment wasinserted into the plasmid pUCT at the EcoRI-SacI site (downstream of thelac promoter) to give a recombinant plasmid pTBH.

EXAMPLE 6 Reduction in GC Ratio in BRD Gene

[0113] For further attaining high level expression of the BRD gene inE.coli, a plasmid pTSBH was constructed by substituting a DNA lower inGC ratio for the segment from the 1st to 118th base of the same gene inthe plasmid pTBH constructed in Example 5, without altering the aminoacid sequence coded thereby, as follows.

[0114] A double-stranded DNA having the sequence shown under SEQ ID NO:9in the sequence listing was prepared by the conventional method. Thiswas digested with EcoRI and XhoI, and a plasmid pTSBH substituted forthe DNA fragment detached from pTBH by digestion with the samerestriction enzymes and containing a 5′ terminal portion of the BRD genewas obtained.

EXAMPLE 7 Construction of Recombinant Plasmid Containing both BRD Geneand Glucose Dehydrogenase Gene

[0115] A double-stranded DNA resulting from addition of theE.coli-derived Shine-Dalgarno sequence (9 bases) at 5 bases upstream ofthe initiation codon of the strain Bacillus megaterium IAM 1030-derivedglucose dehydrogenase (hereinafter referred to as GDH) gene, of a SacIcleavage point just before the above sequence and of a BamHI cleavagepoint just behind the termination codon was prepared in the followingmanner. Based on the nucleotide sequence information about the GDH gene,an N-terminal DNA primer resulting from addition of the E.coli-derivedShine-Dalgarno sequence (9 bases) at 5 bases upstream of the initiationcodon of the structural gene of GDH and further addition of a SacIcleavage point just before the above sequence, and a C-terminal DNAprimer resulting from addition of a BamHI site just behind thetermination codon of the structural gene of GDH were synthesized by theconventional method. The nucleotide sequences of these two primers areshown under SEQ ID NO:10 and SEQ ID NO:11, respectively, in the sequencelisting. Using these two DNA primers, together with the plasmid pGDK1(Eur. J. Biochem. 186, 389 (1989)) as the template, a double-strandedDNA was synthesized by PCR. The DNA fragment obtained was digested withSacI and BamHI and inserted into the SacI-BamHI site (occurringdownstream of the BRD gene) of the pTSBH constructed in Example 5 togive a recombinant plasmid pTSBG1. The method of constructing pTSBG1 andthe structure thereof are shown in FIG. 2.

EXAMPLE 8 Production of Recombinant E.coli

[0116]E.coli HB101 (product of Takara Shuzo) was transformed using therecombinant plasmids pTBH, pSTBH and pTSBG1 obtained in Examples 5, 6and 7, to give recombinant E.coli HB101 (pTBH), HB101 (pTSBH) and HB101(pTSBG1), respectively. Among the thus-obtained transformants, E.coliHB101 (pTSBH) and HB101 (pTSBG1) have been deposited with the NationalInstitute of Advanced Industrial Science and Technology InternationalPatent Organism Depositary (Address: Central 6, 1-1 Higashi 1-chome,Tsukuba City, Ibaraki Prefecture, Japan) under the accession number FERMBP-7118 (deposition date: Apr. 11, 2000) and the accession number FERMBP-7119 (deposition date: Apr. 11, 2000), respectively.

[0117] Further, the plasmid pGDA2 (J. Biol. Chem., (1989), 264, 6381)was double-digested with EcoRI and PstI and the thus-obtained DNAfragment (about 0.9 kb) containing the Bacillus megaterium IWG3-derivedGDH gene was inserted into the plasmid pSTV28 (product of Takara Shuzo)at the EcoRI-PstI site thereof to construct a recombinant plasmid pSTVG.E.coli HB101 (pTSBH) made competent in advance by the calcium chloridemethod was transformed with this plasmid pSTVG at a high rate ofintroduction. Thus, E.coli HB101 (pTSBH, pSTVG) was obtained with ease.

EXAMPLE 9 BRD Expression in Recombinant E.coli

[0118] The recombinants E.coli HB101 (pTBH) and HB101 (pTSBH) obtainedin Example 8 were each shake-cultured on 2×YT medium containing 200μg/ml of ampicillin at 28° C. for 15 hours. A 1-ml portion of thispreculture fluid was inoculated into 100 ml of a medium sterilized byautoclaving in a 500-ml Sakaguchi flaks and comprising 1.5% (w/v)glycerol, 1.5% (w/v) Bacto tryptone, 0.4% (w/v) Bacto yeast extract,0.2% (w/v) sodium chloride, 0.8% (w/v) potassium dihydrogen phosphate,0.05% (w/v) magnesium sulfate heptahydrate, and 0.033% (w/v) AdekanolLG109 (product of Asahi Denka Kogyo), as adjusted to pH 6.0, and shakeculture was carried out at 30° C. for 60 hours. Cells were harvestedfrom such culture fluids by using a centrifuge, then suspended in 100 mMphosphate buffer (pH 6.5) and ultrasonically disrupted to give acell-free extract.

[0119] The BRD activity of this cell-free extract was determined in thefollowing manner. Thus, the BRD activity was determined by adding thesubstrate N-benzyl-3-pyrrolidinone (1 mM), the coenzyme NADPH (0.167 mM)and the enzyme to 100 mM phosphate buffer (pH 6.5) and measuring thedecrease in absorbance at the wavelength 340 nm at 30° C. The enzymeactivity capable of oxidizing 1 μmol of NADPH to NADP in 1 minute underthese reaction conditions was defined as 1 unit. Thus-determined BRDactivities of the cell-free extracts were expressed in terms of specificactivity and compared with that of the transformant holding the vectorplasmid pUCNT. Comparison was also made with the BRD activity of acell-free extract derived from the strain Micrococcus luteus IFO 13867as prepared in the same manner as in Example 1. The results thusobtained are shown in Table 2. TABLE 2 Specific BRD activity Name ofstrain (U/mg) E. coli HB101(pUCNT) <0.01 E. coli HB101(pTBH) 0.06 E.coli HB101(pTSBH) 0.61 Micrococcus luteus IFO 13867 0.06

[0120] As for E.coli HB101 (pTSBH), a distinct increase in BRD activitywas observed as compared with E.coli HB101 (pUCNT) which is transformedwith the vector plasmid alone and, when compared with the strainMicrococcus luteus IFO 13867, the activity was about 10-fold higher.

EXAMPLE 10 Simultaneous Expression of BRD and GDH in Recombinant E.coli

[0121] The recombinant E.coli HB101 (pTSBG1) and HB101 (pTSBH, pSTVG)obtained in Example 8 were cultured and treated in the same manner as inExample 9 to give the respective cell-free extracts, which were assayedfor GDH activity in the following manner. The GDH activity wasdetermined by adding the substrate glucose (0.1 M), the coenzyme NADP (2mM) and the enzyme to 1 M Tris hydrochloride buffer (pH 8.0) andmeasuring the increase in absorbance at the wavelength 340 nm at 25° C.The enzyme activity capable of reducing 1 μmol of NADP to NADPH in 1minute under these reaction conditions was defined as 1 unit. The BRDactivity was also determined in the same manner as in Example 9.Thus-determined BRD and GDH activities of the cell-free extracts wereeach expressed in terms of specific activity and compared with those ofE.coli HB101 (pTSBH) and HB101 (pUCNT) which is transformed with thevector alone. The results are shown in Table 3. TABLE 3 Specific BRDactivity Specific GDH activity Name of strain (U/mg) (U/mg) E. coliHB101(pUCNT) <0.01 <0.01 E. coli HB101(pTSBH) 0.61 <0.01 E. coliHB101(pTSBG1) 0.52 89 E. coli HB101(pTSBH, 0.69 3.2 pSTVG)

[0122] As for E.coli HB101 (pTSBG1) and HB101 (pTSBH, pSTVG), distinctincreases in BRD activity and GDH activity were observed as comparedwith E.coli HB101 (pUCNT) which is transformed with the vector plasmidalone.

EXAMPLE 11 Synthesis of (S)-N-benzyl-3-pyrrolidinol fromN-benzyl-3-pyrrolidinone using Recombinant E.coli Produced byIntroduction of BRD Gene

[0123] The culture fluid of the recombinant E.coli HB101 (pTSBH)obtained in Example 9 was ultrasonically disrupted using SONIFIRE 250(product of BRANSON). To 25 ml of this cell disruption fluid were added1,350 U of glucose dehydrogenase (product of Amano Pharmaceutical), 3.0g of glucose, 3.0 mg of NADP and 0.25 g of N-benzyl-3-pyrrolidinone.While this reaction mixture was stirred at 30° C. with pH adjusted to6.5 using 5 M hydrochloric acid or sodium hydroxide,N-benzyl-3-pyrrolidinone was added thereto at an interval of 0.25g/hour. After addition of a total of 2.0 g of N-benzyl-3-pyrrolidinone,stirring was further continued for 20 hours. After completion of thereaction, 2.5 ml of a 5 M aqueous solution of sodium hydroxide wasadded, the mixture was extracted with toluene, and the solvent wasremoved. Analysis of the resulting extract revealed thatN-benzyl-3-pyrrolidinol was obtained in 74% yield. TheN-benzyl-3-pyrrolidinol produced on that occasion was the S form with anoptical purity of not less than 99% ee.

[0124] The quantity of N-benzyl-3-pyrrolidinone andN-benzyl-3-pyrrolidinol was determined by gas chromatography (column:Uniport B 10% PEG-20 M (3.0 mm ID×1.0 m), column temperature: 200° C.,carrier gas: nitrogen, detection: FID). The optical purity of(S)-N-benzyl-3-pyrrolidinol was determined by high performance liquidchromatography (column: Chiralcel OB (product of Daicel ChemicalIndustries), eluent: n-hexane/isopropanol/diethylamine=950/50/1, flowrate: 1 ml/min, detection: 254 nm).

EXAMPLE 12 Synthesis of (S)-N-benzyl-3-pyrrolidinol fromN-benzyl-3-pyrrolidinone using Recombinant E.coli Capable ofSimultaneous Expression of BRD and Glucose Dehydrogenase

[0125] To 25 ml of the culture fluid of the recombinant E.coli HB101(pTSBG1) obtained in Example 9 were added 2.5 g of glucose, 3.0 mg ofNADP and 0.25 g of N-benzyl-3-pyrrolidinone. While this reaction mixturewas stirred at 30° C. with pH adjusted to 6.5 using 5 M hydrochloricacid or sodium hydroxide, N-benzyl-3-pyrrolidinone was added thereto atan interval of 0.25 g/2 hours. After addition of a total of 1.0 g ofN-benzyl-3-pyrrolidinone, stirring was further continued for 17 hours.After completion of the reaction, 1.2 ml of a 5 M aqueous solution ofsodium hydroxide was added, the mixture was extracted with toluene, andthe solvent was removed. Analysis of the resulting extract revealed thatN-benzyl-3-pyrrolidinol was obtained in 92% yield. TheN-benzyl-3-pyrrolidinol produced on that occasion was the S form with anoptical purity of not less than 99% ee.

EXAMPLE 13 Synthesis of (S)-N-benzyl-3-pyrrolidinol fromN-benzyl-3-pyrrolidinone using Recombinant E.coli Capable ofSimultaneous Expression of BRD and Glucose Dehydrogenase

[0126] To 25 ml of the culture fluid of the recombinant E.coli HB101(pTSBH, pSTVG) obtained in Example 9 were added 2.5 g of glucose, 3.0 mgof NADP and 0.25 g of N-benzyl-3-pyrrolidinone. While this reactionmixture was stirred at 30° C. with pH adjusted to 6.5 using 5 Mhydrochloric acid or sodium hydroxide, N-benzyl-3-pyrrolidinone wasadded thereto at an interval of 0.25 g/hour. After addition of a totalof 2.0 g of N-benzyl-3-pyrrolidinone, stirring was further continued for16 hours. After completion of the reaction, 2.5 ml of a 5 M aqueoussolution of sodium hydroxide was added, the mixture was extracted withtoluene, and the solvent was removed. Analysis of the resulting extractrevealed that N-benzyl-3-pyrrolidinol was obtained in 93% yield. TheN-benzyl-3-pyrrolidinol produced on that occasion was the S form with anoptical purity of not less than 99% ee.

INDUSTRIAL APPLICABILITY

[0127] As a result of cloning of a gene of a polypeptide having enzymeactivity in asymmetrically reducing N-benzyl-3-pyrrolidinone to produce(S)-N-benzyl-3-pyrrolidinol and analysis of the nucleotide sequencethereof, it has become possible to obtain a transformant capable ofproducing, at high levels, the above polypeptide. It has also becomepossible to obtain a transformant capable of producing, at high levels,the polypeptide and glucose dehydrogenase simultaneously. Furthermore,it has become possible to efficiently synthesize(S)-N-benzyl-3-pyrrolidinol from N-benzyl-3-pyrrolidinone using suchtransformants.

1 11 1 277 PRT Micrococcus luteus 1 Met Arg Arg Met Thr Leu Pro Ser GlyGlu Ser Ile Pro Val Leu Gly 1 5 10 15 Gln Gly Thr Trp Gly Trp Gly GluAsp Pro Gly Arg Arg Gly Asp Glu 20 25 30 Val Ala Ala Leu His Ala Gly LeuGlu Leu Gly Met Thr Leu Val Asp 35 40 45 Thr Ala Glu Met Tyr Ala Asp GlyGly Ala Glu Glu Val Ala Gly Glu 50 55 60 Ala Leu Ala Gly Arg Arg Asp GluAla Phe Val Val Ser Lys Val Met 65 70 75 80 Pro Ser His Ala Ser Arg SerGly Thr Ile Ala Ala Cys Glu Arg Ser 85 90 95 Leu Lys Arg Leu Gly Thr AspArg Ile Asp Leu Tyr Leu Leu His Trp 100 105 110 Gln Gly Arg Tyr Pro LeuGln Asp Thr Val Ala Ala Phe His Gln Leu 115 120 125 Val Glu Asp Gly LysIle Arg Tyr Trp Gly Val Ser Asn Phe Asp His 130 135 140 Arg Ala Leu AlaGlu Leu Gln Asp Val Pro Gly Thr Ser Gly Leu Thr 145 150 155 160 Thr AspGln Val Leu Tyr Asn Leu Ser Arg Arg Gly Pro Glu Tyr Asp 165 170 175 LeuLeu Pro Trp Cys Ala Asp His Gln Leu Pro Val Met Ala Tyr Ser 180 185 190Pro Ile Glu Gln Gly Arg Ile Leu Asp Asp Thr Thr Leu Asn Asp Val 195 200205 Ala Ala Arg His Ser Val Ser Pro Ala Ala Ala Ala Leu Ala Trp Val 210215 220 Leu Arg Arg Asp Ser Leu Cys Thr Ile Pro Lys Ala Ser Ser Pro Gln225 230 235 240 His Val Arg Asp Asn Ala Thr Ala Leu Asp Val Glu Leu ThrArg Glu 245 250 255 Asp Leu Asp Ala Leu Asp Arg Ala Phe Pro Pro Pro SerGly Pro Arg 260 265 270 Pro Leu Glu Met Leu 275 2 1410 DNA Micrococcusluteus CDS (108)..(938) 2 ggtacccgcc gccctcctat aagccagcac cggtcgaggacgcgccggcc cttcgaggat 60 ctcagcccac gtcccgcctc aggacaacca gaaggaagtgatcgcgg atg cga cgg 116 Met Arg Arg 1 atg acg ctg ccg agt ggg gag tccatc cct gtg ctg ggc cag ggc acc 164 Met Thr Leu Pro Ser Gly Glu Ser IlePro Val Leu Gly Gln Gly Thr 5 10 15 tgg ggc tgg ggt gag gac ccc ggc cgccgc ggc gac gag gtc gcc gcg 212 Trp Gly Trp Gly Glu Asp Pro Gly Arg ArgGly Asp Glu Val Ala Ala 20 25 30 35 ctg cac gcc ggc ctc gag ctg ggc atgacg ctg gtc gac acc gcc gag 260 Leu His Ala Gly Leu Glu Leu Gly Met ThrLeu Val Asp Thr Ala Glu 40 45 50 atg tac gcc gac ggc ggt gcg gag gag gtggct ggt gaa gca ttg gcg 308 Met Tyr Ala Asp Gly Gly Ala Glu Glu Val AlaGly Glu Ala Leu Ala 55 60 65 ggt cgc cgc gac gag gcg ttc gtg gtc agc aaggtc atg ccg tcc cac 356 Gly Arg Arg Asp Glu Ala Phe Val Val Ser Lys ValMet Pro Ser His 70 75 80 gcc tcc cgt tcc ggc acg atc gcg gcc tgc gaa cgcagc ctg aaa cgc 404 Ala Ser Arg Ser Gly Thr Ile Ala Ala Cys Glu Arg SerLeu Lys Arg 85 90 95 ctg ggc acc gat cgg atc gac ctc tac ctg ctg cac tggcag ggc agg 452 Leu Gly Thr Asp Arg Ile Asp Leu Tyr Leu Leu His Trp GlnGly Arg 100 105 110 115 tac ccg ctg cag gac acc gtc gcg gcc ttc cac cagctc gtc gag gac 500 Tyr Pro Leu Gln Asp Thr Val Ala Ala Phe His Gln LeuVal Glu Asp 120 125 130 ggg aaa atc cga tac tgg ggc gtc agc aac ttc gaccac cgg gcc ctc 548 Gly Lys Ile Arg Tyr Trp Gly Val Ser Asn Phe Asp HisArg Ala Leu 135 140 145 gcc gag ctg cag gac gtg ccg ggc acc agc ggg ctgacc acg gat cag 596 Ala Glu Leu Gln Asp Val Pro Gly Thr Ser Gly Leu ThrThr Asp Gln 150 155 160 gtg ctg tac aac ctg tcg cgg cga gga ccg gag tacgac ctg ctg ccg 644 Val Leu Tyr Asn Leu Ser Arg Arg Gly Pro Glu Tyr AspLeu Leu Pro 165 170 175 tgg tgc gcc gac cac cag ctg ccg gtc atg gcg tactcg ccg atc gag 692 Trp Cys Ala Asp His Gln Leu Pro Val Met Ala Tyr SerPro Ile Glu 180 185 190 195 cag ggc cgc atc ctt gac gac acg acg ctg aacgac gtc gcg gcc cgt 740 Gln Gly Arg Ile Leu Asp Asp Thr Thr Leu Asn AspVal Ala Ala Arg 200 205 210 cac agc gtc agc ccc gcg gcg gcg gcc ctt gcctgg gtg ctg cgc cgc 788 His Ser Val Ser Pro Ala Ala Ala Ala Leu Ala TrpVal Leu Arg Arg 215 220 225 gac tcg ctc tgc acg atc ccc aag gcg agc agcccg cag cac gtg cgc 836 Asp Ser Leu Cys Thr Ile Pro Lys Ala Ser Ser ProGln His Val Arg 230 235 240 gac aac gcc aca gca ctg gac gtg gag ctg acccgc gaa gac ctg gat 884 Asp Asn Ala Thr Ala Leu Asp Val Glu Leu Thr ArgGlu Asp Leu Asp 245 250 255 gct ctg gac cgt gcg ttt ccg ccc ccg agc ggaccg cga cca ctg gaa 932 Ala Leu Asp Arg Ala Phe Pro Pro Pro Ser Gly ProArg Pro Leu Glu 260 265 270 275 atg ctg tgaccctgcc ccagggcgca gcccggtcggtccgggcggt ccgggcagtc 988 Met Leu cgggcagcgc tccggtcagc gcaagtctccgaaggacctg cctgtcacct cctcctgaac 1048 ctgtgcacgc catccatcga ctcctttcctcgagccctgt cgggttcgcg gtaggcgctg 1108 atcatccgct ggcaggtccc ccaagtggcctcgagccggg ccctctgctt gtcggtgagc 1168 aacccggttc cggcgtgcag ggttcgacgggcggagtaga gcgggtcgcc cgtgcggccg 1228 cggtggccat gcaggtcctg ctggacccggcggtggcagc ggaccaacgc gtcgccggct 1288 aaccggactg cgagcgaccg gcgttgtggacgcagacgac ctggacactg ggccgtgcgg 1348 tcaggaggat ctccaaagtc ggcggcgggggttcaggcga tgtcgaggaa ggaacggagc 1408 tc 1410 3 20 DNA ArtificialSequence Description of Artificial Sequence Primer 3 gayacngcngaratgtaygc 20 4 20 DNA Artificial Sequence Description of ArtificialSequence Primer 4 tcytcnacna gytgrtgraa 20 5 26 DNA Artificial SequenceDescription of Artificial Sequence Primer 5 gcgcatatgc gacggatgac gctgcc26 6 32 DNA Artificial Sequence Description of Artificial SequencePrimer 6 ggcgaattct tacagcattt ccagtggtcg cg 32 7 46 DNA ArtificialSequence Description of Artificial Sequence Primer 7 gcgaattctaaggagattta tatatgcgac ggatgacgct gccgag 46 8 29 DNA Artificial SequenceDescription of Artificial Sequence Primer 8 caggagctct tacagcatttccagtggtc 29 9 144 DNA Artificial Sequence Description of ArtificialSequence Synthetic double-stranded DNA 9 gaattctaag gagatttacatatgcgtcgt atgactttac catctggtga atctattcca 60 gttttaggtc aaggtacttggggttggggt gaagatccag gtcgtcgtgg tgatgaagtt 120 gctgctttac atgctggtctcgag 144 10 33 DNA Artificial Sequence Description of ArtificialSequence Primer 10 caggagctct aaggaggtta acaatgtata aag 33 11 28 DNAArtificial Sequence Description of Artificial Sequence Primer 11cacggatcct tatccgcgtc ctgcttgg 28

1. A polypeptide having the following physicochemical properties (1) tb(5): (1) Action; It asymmetrically reduces N-benzyl-3-pyrrolidinone toproduce (S)-N-benzyl-3-pyrrolidinol with NADPH as a coenzyme; (2)Optimum action pH: 4.5 to 5.5; (3) Optimum action temperature: 40° C. to45° C.; (4) Molecular weight: About 29,000 as determined by gelfiltration analysis, about 35,000 as determined by SDS-polyacrylamidegel electrophoresis analysis; (5) Inhibitor: It is inhibited by thedivalent copper ion.
 2. A polypeptide described in the following (a) or(b): (a) A polypeptide having the amino acid sequence shown under SEQ IDNO:1 in the sequence listing; (b) A polypeptide having an amino acidsequence obtainable from the amino acid sequence shown under SEQ ID NO:1in the sequence listing by substitution, insertion, deletion and/oraddition of one or more amino acids and having enzyme activity inasymmetrically reducing N-benzyl-3-pyrrolidinone to produce(S)-N-benzyl-3-pyrrolidinol.
 3. The polypeptide according to claim 1 or2 which is derived from a microorganism belonging to the genusMicrococcus.
 4. The polypeptide according to claim 3, wherein saidmicroorganism is the strain Micrococcus luteus IFO
 13867. 5. A DNAcoding for the polypeptide according to any of claims 1 to
 4. 6. A DNAcoding for a polypeptide having enzyme activity in asymmetricallyreducing N-benzyl-3-pyrrolidinone to produce(S)-N-benzyl-3-pyrrolidinol, and hybridizing with a DNA having anucleotide sequence shown under SEQ ID NO:2 in the sequence listingunder stringent conditions.
 7. A DNA coding for a polypeptide havingenzyme activity in asymmetrically reducing N-benzyl-3-pyrrolidinone toproduce (S)-N-benzyl-3-pyrrolidinol, and having at least 60% sequenceidentity with a nucleotide sequence shown under SEQ ID NO:2 in thesequence listing.
 8. An expression vector containing DNAs according toany of claims 5 to
 7. 9. The expression vector according to claim 8,which is a plasmid pTSBH.
 10. The expression vector according to claim8, which contains a DNA coding for a polypeptide having glucosedehydrogenase activity.
 11. The expression vector according to claim 10,wherein said polypeptide having glucose dehydrogenase activity is aBacillus megaterium-derived glucose dehydrogenase.
 12. The expressionvector according to claim 11, which is a plasmid pTSBG1.
 13. Atransformant containing the expression vector according to any of claims8 to
 12. 14. A transformant containing both the expression vectoraccording to claim 8 or 9 and an expression vector containing a DNAcoding for a polypeptide having glucose dehydrogenase activity.
 15. Thetransformant according to claim 14, wherein said polypeptide havingglucose dehydrogenase activity is a Bacillus megaterium-derived glucosedehydrogenase.
 16. The transformant according to any of claims 13 to 15,wherein a host thereof is Escherichia coli.
 17. The transformantaccording to claim 16, which is Escherichia coli HB101 (pTSBH).
 18. Thetransformant according to claim 16, which is Escherichia coli HB101(pTSBG1).
 19. The transformant according to claim 16, which isEscherichia coli HB101 (pTSBH, pSTVG).
 20. A production method of(S)-N-benzyl-3-pyrrolidinol comprising a step of reacting thetransformant according to any of claims 13 to 19 and/or a treatedproduct thereof with N-benzyl-3-pyrrolidinone, and a step of harvestingthe thus-produced (S)-N-benzyl-3-pyrrolidinol.
 21. The method accordingto claim 20, wherein the step of reacting is carried out in the presenceof a coenzyme regenerating system.